The Immunopathology of Pulmonary Rejection after Murine Lung Transplantation.

To improve outcomes following lung transplantation, it is essential to understand the immunological mechanisms that result in chronic graft failure. The associated clinical syndrome is termed chronic lung allograft dysfunction (CLAD), which is known to be induced by alloimmune-dependent (i.e., rejection) and alloimmune-independent factors (e.g., infections, reflux and environmental factors). We aimed to explore the alloimmune-related mechanism, i.e., pulmonary rejection. In this study, we use a murine orthotopic left lung transplant model using isografts and allografts (C57BL/6 or BALB/c as donors to C57BL/6 recipients), with daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone). Serial sacrifice was performed at days 1, 7 and 35 post-transplantation (n = 6 at each time point for each group). Left transplanted lungs were harvested, a single-cell suspension was made and absolute numbers of immune cells were quantified using multicolor flow cytometry. The rejection process followed the principles of a classic immune response, including innate but mainly adaptive immune cells. At day 7 following transplantation, the numbers of interstitial macrophages, monocytes, dendritic cells, NK cells, NKT cells, CD4+ T cells and CD8+ T and B cells were increased in allografts compared with isografts. Only dendritic cells and CD4+ T cells remained elevated at day 35 in allografts. Our study provides insights into the immunological mechanisms of true pulmonary rejection after murine lung transplantation. These results might be important in further research on diagnostic evaluation and treatment for CLAD.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

The effect of multiple outgrowths from bronchial tissue explants on progenitor/stem cell number in primary bronchial epithelial cell cultures from smokers and patients with COPD.

Background: Although studies suggest a deficiency in stem cell numbers in chronic airway diseases such as chronic obstructive pulmonary disease (COPD), the role of bronchial epithelial progenitor/stem (P/S) cells is not clear. The objectives of this study were to investigate expression of progenitor/stem (P/S) cell markers, cytokeratin (CK) 5, CK14 and p63 in bronchial epithelial explants and cell cultures obtained from smokers with and without COPD following multiple outgrowths, and to study this effect on bronchial epithelial cell (BEC) proliferation. Methods: Bronchial epithelial explants were dissected from lung explants and cultured on coverslips. Confluent cultures were obtained after 3-4 weeks' (transfer, Tr1), explants were then transferred and cultured for a second (Tr2) and third (Tr3) time, respectively. At each stage, expression of CK5, CK14 and p63 in explants and BEC were determined by immunostaining. In parallel experiments, outgrowing cells from explants were counted after 4wks, and explants subsequently transferred to obtain new cultures for a further 3 times. Results: As the transfer number advanced, CK5, CK14 and p63 expression was decreased in both explants and BEC from both smokers without COPD and patients with COPD, with a more pronounced decrease in BEC numbers in the COPD group. Total cell numbers cultured from explants were decreased with advancing outgrowth number in both groups. Smoking status and lung function parameters were correlated with reduced P/S marker expression and cell numbers. Conclusion: Our findings suggest that the number of P/S cells in airway epithelium may play a role in the pathogenesis of COPD, as well as a role in the proliferation of airway epithelial cells, in vitro.

Systems toxicology of complex wood combustion aerosol reveals gaseous carbonyl compounds as critical constituents.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air-liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease.

Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification.

Aqp5-/- mice exhibit reduced maximal body O2 consumption under cold exposure, normal pulmonary gas exchange, and impaired formation of brown adipose tissue.

The fundamental body functions that determine maximal O2 uptake (V̇o2max) have not been studied in Aqp5-/- mice (aquaporin 5, AQP5). We measured V̇o2max to globally assess these functions and then investigated why it was found altered in Aqp5-/- mice. V̇o2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o2max reduced in Aqp5-/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5-/- mice. Lung function parameters as well as arterial O2 saturation were normal and identical between Aqp5-/- and WT mice, indicating that AQP5 does not contribute to pulmonary O2 exchange. The cause for the decreased V̇o2max thus might be found in decreased O2 consumption of an intensely O2-consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5-/- animals. We conclude that lack of AQP5 does not affect pulmonary O2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o2max under this condition.

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

A transcriptomics-guided drug target discovery strategy identifies receptor ligands for lung regeneration.

Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here, we used a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors, of which we screened the function in lung organoids. We found several drug targets with regenerative potential, of which EP and IP prostanoid receptor ligands had the most profound therapeutic potential in restoring cigarette smoke-induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we found, using single-cell RNA sequencing analysis, that circadian clock and cell cycle/apoptosis signaling pathways were differentially expressed in alveolar epithelial progenitor cells in patients with COPD and in a relevant model of COPD, which was prevented by prostaglandin E2 or prostacyclin mimetics. We conclude that specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.

Endolysosomal Cation Channels and Lung Disease.

Endolysosomal cation channels are emerging as key players of endolysosomal function such as endolysosomal trafficking, fusion/fission, lysosomal pH regulation, autophagy, lysosomal exocytosis, and endocytosis. Diseases comprise lysosomal storage disorders (LSDs) and neurodegenerative diseases, metabolic diseases, pigmentation defects, cancer, immune disorders, autophagy related diseases, infectious diseases and many more. Involvement in lung diseases has not been a focus of attention so far but recent developments in the field suggest critical functions in lung physiology and pathophysiology. Thus, loss of TRPML3 was discovered to exacerbate emphysema formation and cigarette smoke induced COPD due to dysregulated matrix metalloproteinase 12 (MMP-12) levels in the extracellular matrix of the lung, a known risk factor for emphysema/COPD. While direct lung function measurements with the exception of TRPML3 are missing for other endolysosomal cation channels or channels expressed in lysosome related organelles (LRO) in the lung, links between those channels and important roles in lung physiology have been established such as the role of P2X4 in surfactant release from alveolar epithelial Type II cells. Other channels with demonstrated functions and disease relevance in the lung such as TRPM2, TRPV2, or TRPA1 may mediate their effects due to plasma membrane expression but evidence accumulates that these channels might also be expressed in endolysosomes, suggesting additional and/or dual roles of these channels in cell and intracellular membranes. We will discuss here the current knowledge on cation channels residing in endolysosomes or LROs with respect to their emerging roles in lung disease.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Cathepsin C inhibition as a potential treatment strategy in cancer.

Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

Cathepsin B promotes collagen biosynthesis, which drives bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major complication after lung transplantation (LTx). BOS is characterised by massive peribronchial fibrosis, leading to air trapping-induced pulmonary dysfunction. Cathepsin B, a lysosomal cysteine protease, has been shown to enforce fibrotic pathways in several diseases. However, the relevance of cathepsin B in BOS progression has not yet been addressed. The aim of the study was to elucidate the function of cathepsin B in BOS pathogenesis.We determined cathepsin B levels in bronchoalveolar lavage fluid (BALF) and lung tissue from healthy donors (HD) and BOS LTx patients. Cathepsin B activity was assessed via a fluorescence resonance energy transfer-based assay and protein expression was determined using Western blotting, ELISA and immunostaining. To investigate the impact of cathepsin B in the pathophysiology of BOS, we used an in vivo orthotopic left LTx mouse model. Mechanistic studies were performed in vitro using macrophage and fibroblast cell lines.We found a significant increase of cathepsin B activity in BALF and lung tissue from BOS patients, as well as in our murine model of lymphocytic bronchiolitis. Moreover, cathepsin B activity was associated with increased biosynthesis of collagen and had a negative effect on lung function. We observed that cathepsin B was mainly expressed in macrophages that infiltrated areas characterised by a massive accumulation of collagen deposition. Mechanistically, macrophage-derived cathepsin B contributed to transforming growth factor-ß1-dependent activation of fibroblasts, and its inhibition reversed the phenotype.Infiltrating macrophages release active cathepsin B, thereby promoting fibroblast activation and subsequent collagen deposition, which drive BOS. Cathepsin B represents a promising therapeutic target to prevent the progression of BOS.

Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

CARM1 regulates senescence during airway epithelial cell injury in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung disease. Although cigarette smoke was considered the main cause of development, the heterogeneous nature of the disease leaves it unclear whether other factors contribute to the predisposition or impaired regeneration response observed. Recently, epigenetic modification has emerged to be a key player in the pathogenesis of COPD. The addition of methyl groups to arginine residues in both histone and nonhistone proteins by protein arginine methyltransferases (PRMTs) is an important posttranslational epigenetic modification event regulating cellular proliferation, differentiation, apoptosis, and senescence. Here, we hypothesize that coactivator-associated arginine methyltransferase-1 (CARM1) regulates airway epithelial cell injury in COPD pathogenesis by controlling cellular senescence. Using the naphthalene (NA)-induced mouse model of airway epithelial damage, we demonstrate that loss of CC10-positive club cells is accompanied by a reduction in CARM1-expressing cells of the airway epithelium. Furthermore, Carm1 haploinsuffficent mice showed perturbed club cell regeneration following NA treatment. In addition, CARM1 reduction led to decreased numbers of antisenescent sirtuin 1-expressing cells accompanied by higher p21, p16, and ß-galactosidase-positive senescent cells in the mouse airway following NA treatment. Importantly, CARM1-silenced human bronchial epithelial cells showed impaired wound healing and higher ß-galactosidase activity. These results demonstrate that CARM1 contributes to airway repair and regeneration by regulating airway epithelial cell senescence.

The Notch ligand DNER regulates macrophage IFNγ release in chronic obstructive pulmonary disease.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide with no curative therapy. A non-canonical Notch ligand, DNER, has been recently identified in GWAS to associate with COPD severity, but its function and contribution to COPD is unknown. METHODS: DNER localisation was assessed in lung tissue from healthy and COPD patients, and cigarette smoke (CS) exposed mice. Microarray analysis was performed on WT and DNER deficient M1 and M2 bone marrow-derived macrophages (BMDM), and gene set enrichment undertaken. WT and DNER deficient mice were exposed to CS or filtered air for 3 day and 2 months to assess IFNγ-expressing macrophages and emphysema development. Notch and NFKB active subunits were quantified in WT and DNER deficient LPS-treated and untreated BMDM. FINDINGS: Immunofluorescence staining revealed DNER localised to macrophages in lung tissue from COPD patients and mice. Human and murine macrophages showed enhanced DNER expression in response to inflammation. Interestingly, pro-inflammatory DNER deficient BMDMs exhibited impaired NICD1/NFKB dependent IFNγ signalling and reduced nuclear NICD1/NFKB translocation. Furthermore, decreased IFNγ production and Notch1 activation in recruited macrophages from CS exposed DNER deficient mice were observed, protecting against emphysema and lung dysfunction. INTERPRETATION: DNER is a novel protein induced in COPD patients and 6 months CS-exposed mice that regulates IFNγ secretion via non-canonical Notch in pro-inflammatory recruited macrophages. These results provide a new pathway involved in COPD immunity that could contribute to the discovery of innovative therapeutic targets. FUNDING: This work was supported from the Helmholtz Alliance 'Aging and Metabolic Programming, AMPro'.

A Drosophila model of cigarette smoke induced COPD identifies Nrf2 signaling as an expedient target for intervention.

Chronic obstructive pulmonary disease (COPD) is among the most important causes of death. Signaling systems that are relevant for tissue repair and detoxification of reactive oxygen species or xenobiotics are thought to be impaired in lungs of patients suffering from this disease. Here, we developed a simple cigarette smoke induced Drosophila model of COPD based on chronic cigarette smoke exposure that recapitulates major pathological hallmarks of the disease and thus can be used to investigate new therapeutic strategies. Chronic cigarette smoke exposure led to premature death of the animals and induced a set of phenotypes reminiscent of those seen in COPD patients, including reduced physical activity, reduced body fat, increased metabolic rate and a substantial reduction of the respiratory surface. A detailed transcriptomic analysis revealed that especially the TGF-ß, Nrf2 and the JAK/STAT signaling pathways are altered by chronic cigarette smoke exposure. Based on these results, we focused on Nrf2 signaling. A pharmacological intervention study performed with oltipraz, an activator of Nrf2 signaling, increased survival of cigarette smoke exposed animals significantly. Thus, the Drosophila COPD model recapitulates many major hallmarks of COPD and it is highly useful to evaluate the potential of alternative therapeutic strategies.

TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca2+ ([Ca2+]i) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca2+]i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca2+]i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease.

The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus-associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD In both COPD patients and cigarette smoke (CS)-exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS-induced B-cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS-induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS-induced emphysema in vivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol-dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.